1. Field of the Invention
The present invention relates to a method of producing lysergic acid by genetically modifying a fungus. A method for producing dihydroergot alkaloids (dihydrolysergic acid and dihydrolysergol) and lysergol are also provided. This invention discloses the heterologous expression of lysergic acid and novel ergot alkaloids in Aspergillus fumigatus. 
2. Description of the Background Art
Ergot alkaloids derived from lysergic acid have impacted human health for millennia, initially as toxins and more recently as pharmaceuticals; however, important aspects of ergot alkaloid biosynthesis remain unsolved. Ergot alkaloids are pharmaceutically and agriculturally relevant secondary metabolites synthesized by several species of fungi. Historically, ergot alkaloids caused periodic mass human poisonings due to infection of grain crops by the ergot fungus Claviceps purpurea (Matossian, 1989). Agriculturally, ergot alkaloids in forage grasses colonized by endophytic Epichloë spp. [including many fungi recently realigned from genus Neotyphodium (Leuchtmann et al., 2014)] continue to reduce weight gain and fitness in grazing animals (Schardl et al., 2012; Panaccione et al., 2014). Clinically, the structural similarities of ergot alkaloids to monoamine neurotransmitters allow them to treat cognitive and neurological maladies including dementia, migraines, and Parkinson's disease in addition to endocrine disorders such as type 2 diabetes and hyperprolactinemia (e.g., Baskys and Hau, 2007; Morren and Galvez-Jimenez, 2010; Perez-Lloret and Rascol, 2010; Winblad et al., 2008) (see ergot chart below). Indeed, the neurotransmitter-mimicking activities of ergot alkaloids are most infamously evident in the psychoactive drug LSD, a semisynthetic lysergic acid derivative (Hoffman, 1980). Several of the more important pharmaceutical ergot alkaloids are semi-synthetic dihydroergot alkaloids (dihydro prefix abbreviated as DH in subsequent text); natural DHergot alkaloids producers exist, but the genetic basis for their biosynthesis is unknown. In some embodiments of the invention, controlling the ergot alkaloid pathway will facilitate metabolic engineering strategies to produce libraries of ergot derivatives with potentially altered pharmacology. Moreover, by understanding different branches of the ergot alkaloid pathway, we will be able to prepare alternate starting materials for more efficient pharmaceutical synthesis.
Examples of pharmaceutical ergot alkaloids and their uses and derivations1Ergot AlkaloidCurrent semisyntheticEfficient semisynthetic(brand name)Clinical use(s)derivationderivationNicergolineSenile dementia,From LA via other ergotFrom DHlysergol,(Sermion)Alzheimer's,alkaloidslysergol, or DHLAcerebral thrombosisCabergolineHyperprolactinemia,From LA via other ergotFrom DHLA(Caberlin,pituitary prolactinomasalkaloidsDostinex)PergolideParkinson's (elsewhere,From LA via other ergotFrom DHlysergol,(Permax)withdrawn in USA, 2007)alkaloidslysergol, or DHLABromocriptineType 2 diabetes,From α-ergocryptine or(Parlodel,Parkinson's,LA via other ergotCycloset)hyperprolactinemiaalkaloidsErgoloid mesylatesSenile dementiaFrom ergopeptines orFrom DHergopeptines(Hydergine)LA via other ergotor DHLAalkaloidsDHergotamineMigrainesFrom ergotamine or LAFrom DHergotamine(DHE 45,via other ergot alkaloidsor DHLAMigranal)1Abbreviations: LA, lysergic acid; DH, dihydro (meaning lacking a double bond in fourth ring of ergoline nucleus)
Lysergic acid that is used for pharmaceutical production is presently synthesized in one of two methods know by those skilled in the art generally. The first known method involves growing crops of rye that are later infected with an ergot alkaloid producing fungus Claviceps purpurea. During infection, C. purpurea produces structures called sclerotia in place of the native rye grains. The sclerotia contain complex alkaloids that are derived from lysergic acid. At the flowering stage of the rye, the fungus (which has been grown for 5-6 weeks in culture) is inoculated onto the flowers of the grass. Depending on weather conditions, the sclerotia can be harvested after 4-6 weeks. Total ergot alkaloids must be extracted from the sclerotia. All the alkaloids must then be hydrolyzed in a strong base to produce lysergic acid. The second known method is to grow mutant strains of either C. purpurea or Claviceps paspali in either stationary surface cultures or submerged cultures-all containing a growth medium. There are three cultivation steps: preinoculating tanks, seed tanks, and production fermenters, each requiring a different growth medium. The cultures are grown for several weeks. Our experiences have optimum alkaloid production after 7 weeks of growth. From our experience, alkaloid production is not guaranteed in this method. Similar to the first known method, total complex alkaloids must be extracted and hydrolyzed in this second known method to produce lysergic acid before purification of lysergic acid. The following publications describe the generalities of these known methods of producing lysergic acid: (1) Annis, S. L., and Panaccione, D. G. 1998. Presence of peptide synthetase gene transcripts and accumulation of ergopeptines in Claviceps purpurea and Neotyphodium coenophialum. Canadian Journal of Microbiology 44:80-86; (2) Coyle, C. M., Cheng, J. Z., O'Connor, S. E., Panaccione, D. G. 2010. An old yellow enzyme gene controls the branch point between Aspergillus fumigatus and Claviceps purpurea ergot alkaloid pathways. Applied and Environmental Microbiology 76:3 898-3903; and (3) Kren, V., and Cvak, L. 1999. Ergot, The Genus Claviceps. Harwood Academic Publishers, Amsterdam, page 518.
Unlike the known methods of producing lysergic acid as described above, the present invention provides an efficient method of producing lysergic acid and its purification directly without the need to utilize complex alkaloids.